Post-translational modifications of proliferating cell nuclear antigen (PCNA) are essential for maintaining genome stability. PCNA ubiquitylation facilitates translesion synthesis of damaged DNA templates, and PCNA SUMOylation prevents aberrant recombination. This proposal focuses on understanding the protein interactions regulated by post-translational modifications of PCNA and how these interactions modulate translesion synthesis and recombination. The proposed studies are possible because of a highly innovative technological development made by our group which allows for the production of large quantities of modified PCNA using a split/fusion strategy. Using this approach, we determined the X-ray crystal structures of both ubiquitin-modified PCNA (Ubi-PCNA) and SUMO-modified PCNA (SUMO-PCNA). Aim 1 is to study the interaction and regulation of target proteins by Ubi-PCNA and SUMO-PCNA. Modified PCNA will be analyzed in single-molecule binding and reconstituted enzyme assays. These studies will test the tool belt model of PCNA action. Aim 2 is to study the structure and dynamics of Ubi-PCNA and SUMO-PCNA. Structural studies will determine the dynamics of modified PCNA and test the hypothesis that dynamic protein-protein partner complexes are critical for regulation. Studies will also determine how post-translational modification of PCNA effects interactions with anti-recombinogenic helicases and modulates recombination. Aim 3 is to study the regulation of DNA synthesis and recombination by Ubi-PCNA and SUMO-PCNA. The split/fusion methodology allows expression of constitutively modified PCNA in cells. This allows in vivo analysis of the effects of ubiquitylated and SUMOylated PCNA. These studies will provide a clear understanding of exactly how PCNA recruits proteins to replication forks, how the activities of different proteins bound to PCNA are regulated and coordinated, how the hand-off between different proteins occurs on PCNA during multi-step processes, and how post-translational modifications of PCNA have such different effects on these processes.